DNA encoding a mast cell-derived membrane protein

ABSTRACT

An originally developed efficient signal sequence trapping method was used to screen a cDNA library prepared from cultured mast cells derived from mouse bone marrow. As a result, genes encoding type I membrane proteins and comprising a single immunoglobulin domain in the extracellular domain and a motif for transmitting an inhibitory signal into cells were successfully isolated.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/JP2003/013921, filed Oct. 30, 2003, which claims the benefit ofJapanese Patent Applications Serial No. 2002-316680, filed on Oct. 30,2002, and 2002-354165, filed on Dec. 5, 2002. The contents of allapplications are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to novel membrane proteins derived frommast cells and genes encoding them, as well as methods for producingthem and uses thereof.

BACKGROUND ART

Mast cells are known to act as effector cells in allergic diseases suchas atopic dermatitis, rhinitis, and asthma by releasinginflammation-associated substances such as histamine upon antigenstimulation. Antihistamines or steroids that suppress the production orrelease of inflammation-associated substances from mast cells, or drugsthat antagonize the effect of such substances, are currently being usedfor the treatment of these allergic diseases. However, the developmentof effective drugs with higher selectivity is being anticipated.

Molecules such as FcγRIIB, gp49B, and SIRPα have been known ascandidates of membrane proteins participating in the regulation of mastcell signal transduction. However, the whole picture of the mechanismthat regulates responses to antigen stimulation associated with allergicdiseases remains unclear.

DISCLOSURE OF THIS INVENTION

The present invention provides novel membrane proteins thought to play arole in the regulation of mast cell signal transduction, genes encodingthem, as well as methods for producing them and uses thereof. Themembrane proteins of this invention would be useful for elucidating themechanisms that regulate responses to antigen stimulation, ortransduction of survival or proliferation signals in mast cells.

In order to solve the above problems, the present inventors prepared acDNA library from mouse bone marrow-derived cultured mast cells, a wellcharacterized mast cell model. They then screened this library formolecules containing signal peptides (von Heijne J. Mol. Biol. 184:99-105 (1985)) using an efficient signal sequence trapping system (theSST-REX method) (Kojima T. and Kitamura T. Nature Biotechnol. 17:487-490 (1999)), which was developed by the inventors themselves using aretrovirus-mediated expression cloning system (Kitamura T. et al. Proc.Natl. Acad. Sci. USA 92: 9146-9150 (1995)). In the SST-REX method, alibrary expressing fusion proteins with the constitutively active formof cytokine receptor MPL is screened to look for a cDNA(s) encoding aprotein(s) capable of inducing the cell surface expression of the MPL.As the index used in the method is the acquisition of autonomousproliferation capability in IL-3 dependent cell lines as a result of MPLexpression on the cell surface, a clone of interest can be easilyselected.

After screening 2.0×10⁶ clones, a gene encoding a type I membraneprotein and having a single immunoglobulin domain in the extracellulardomain and a motif for transmitting an inhibitory signal into cells wasidentified. The protein was named MC-PIR1 (later renamed as LMIR1). Inaddition, a molecule whose amino acid sequence showed approximately 90%homology with the immunoglobulin domain of MC-PIR1 was isolated from thesame library, and named MC-PIR2 (later renamed as LMIR2).

These genes had an expression profile specific to mast cells.Furthermore, MC-PIR1 was phosphorylated upon cross-linking, and wascapable of binding to the phosphotyrosine phosphatases SHP-1 and SHP-2,and phosphoinositide phosphatase SHIP, which are adaptor proteinsinvolved in the regulation of signal transduction. MC-PIR2 was found toform a complex with DAP10, DAP12, and FcRγ, ITAM-comprising signalingmolecules. Therefore, these proteins are likely to be membrane proteinsparticipating in the regulation of mast cell signal transduction.

MC-PIR1 and MC-PIR2 genes are derived from mouse. A DNA search usingtheir nucleotide sequences identified human gene homologues CMRF-35H,Irp60, and CMRF-35A. Since these human genes were not known toparticipate in the regulation of mast cell signal transduction, theinformation obtained through MC-PIR1 and MC-PIR2 provides novel uses forthe gene products of these human genes.

Natural ligands for MC-PIR1, MC-PIR2, and the human homologues thereofhave not been identified, but the use of these gene products wouldfacilitate the discovery of these natural ligands. Similarly, the geneproducts may also be useful in screenings for compounds that mimic thefunction of natural ligands, or antibodies. Such compounds orantibodies, obtained by the above screenings, have the potential toinhibit the transduction of the mast cell activation signal, and thus beanti-allergy drugs with a new mechanism of action.

The present invention relates to mast cell-derived membrane proteins,gene encoding them, and molecules functionally equivalent to these, aswell as methods for producing them, and uses thereof. More specifically,the present invention provides:

-   (1) a DNA according to any one of the following (a) to (d):    -   (a) a DNA encoding a protein comprising the amino acid sequence        of SEQ ID NO: 2 or 4,    -   (b) a DNA comprising the coding region of the nucleotide        sequence of SEQ ID NO: 1 or 3,    -   (c) a DNA encoding a protein comprising an amino acid sequence        in which one or more amino acids in the amino acid sequence of        SEQ ID NO: 2 or 4 have been replaced, deleted, inserted, and/or        added,    -   (d) a DNA capable of hybridizing with a DNA comprising the        nucleotide sequence of SEQ ID NO: 1 or 3 under stringent        conditions;-   (2) the DNA of (1) encoding a protein capable of binding to a    protein selected from the group consisting of SHP-1 protein, SHP-2    protein, SHIP protein, DAP10 protein, DAP12 protein, and FcRγ    protein;-   (3) a protein encoded by the DNA of (1);-   (4) a vector into which the DNA of (1) has been inserted;-   (5) a host cell carrying the DNA of (1), or the vector of (4);-   (6) a method for producing the protein of (3), which comprises the    steps of culturing the host cell of (5), and recovering an expressed    protein from said host cell or the culture supernatant thereof;-   (7) an antibody that binds to the protein of (3);-   (8) a polynucleotide comprising at least 15 nucleotides that is    complementary to a DNA comprising the nucleotide sequence of SEQ ID    NO: 1 or 3, or the complementary strand thereof;-   (9) a method of screening for a compound that binds to the protein    of (3), which comprises the following steps of:    -   (a) contacting said protein with a test sample,    -   (b) detecting the binding activity between said protein and said        test sample, and    -   (c) selecting a compound capable of binding to said protein;-   (10) a method of screening for a compound capable of inhibiting the    binding between the protein of (3) and a protein selected from the    group consisting of SHP-1 protein, SHP-2 protein, SHIP protein,    DAP10 protein, DAP12 protein, and FcRγ protein, which comprises the    following steps of:    -   (a) contacting the protein of (3) with a protein selected from        said group in the presence of a test sample,    -   (b) detecting the binding activity between said proteins, and    -   (c) selecting a compound capable of reducing the binding        activity between said proteins compared to that detected in the        absence of said test sample;-   (11) a method for producing an anti-allergy drug, which comprises    the step of mixing the antibody of (7), or a compound obtained using    the method of (9) or (10), with a pharmacologically acceptable    carrier or vehicle.

The present invention provides DNAs encoding novel membrane proteinsderived from mast cells, which are thought to participate in theregulation of the signal transduction in mast cells.

Using a recently established novel signal sequence trapping method (theST-REX method), the present inventors searched a cDNA library preparedfrom mouse bone marrow-derived cultured mast cells, and identified twogenes, each of which encodes a type I membrane protein and comprises asingle immunoglobulin domain in the extracellular domain and a motif fortransmitting an inhibitory signal into cells. The nucleotide sequence ofthe gene named MC-PIR1 is shown in SEQ ID NO: 1. The amino acid sequenceof a protein encoded by the gene is shown in SEQ ID NO: 2. In addition,the nucleotide sequence of the gene named MC-PIR2, having approximately90% homology at the amino acid level with the immunoglobulin domain ofMC-PIR1, is shown in SEQ ID NO: 3, and the amino acid sequence of aprotein encoded by the gene is shown in SEQ ID NO: 4. These genes areexpressed specifically in mast cells. In addition, MC-PIR1 isphosphorylated in response to antigen stimulation, and is capable ofbinding to phosphotyrosine phosphatases SHP-1 and SHP-2 andphosphoinositide phosphatase SHIP, which are adaptor proteinsparticipating in the regulation of signal transduction. On the otherhand, MC-PIR2 is capable of forming a complex with DAP10, DAP12, andFcRγ, which are signaling molecules comprising ITAM. Thus, theseproteins of the present invention are considered to be membrane proteinsparticipating in the regulation of mast cell signal transduction. Suchproteins are expected to be useful, for example, for the development ofanti-allergy drugs having a novel mechanism of action through inhibitingthe transduction of the activation signal in mast cells.

In addition, the present invention comprises a protein functionallyequivalent to a protein encoded by MC-PIR1 DNA or MC-PIR2 DNA(comprising the nucleotide sequence according to SEQ ID NO: 1 or 3).Such proteins include, for example, mutants of these proteins, theirhomologues in organisms other than mouse, and so on. Herein, the phrase“functionally equivalent” means that the protein of interest has abiological or biochemical activity similar to the MC-PIR1 and MC-PIR2proteins. For example, such an activity may be the ability to undergophosphorylation in response to antigen stimulation and bind to adaptorproteins involved in the regulation of signal transduction such asphosphotyrosine phosphatase SHP-1 and SHP-2 and phosphoinositidephosphatase SHIP, or the ability to bind to signaling moleculescomprising ITAM such as DAP10, DAP12, and FcRγ.

To prepare a protein functionally equivalent to another protein, methodsfor introducing mutations into proteins are well known to those skilledin the art. For example, those skilled in the art can prepare a proteinfunctionally equivalent to the MC-PIR1 or MC-PIR2 protein (comprisingthe amino acid sequence of SEQ ID NO: 2 or 4) by introducing anappropriate mutation into an amino acid(s) of the protein usingsite-directed mutagenesis (Hashimoto-Gotoh T. et al. Gene 152:271-275(1995); Zoller M. J. and Smith M. Methods Enzymol. 100:468-500(1983); Kramer W. et al. Nucleic Acids Res. 12: 9441-9456 (1984);Kramer W. and Fritz H. J. Methods Enzymol. 154: 350-367(1987); Kunkel T.A. Proc. Natl. Acad. Sci. USA 82: 488-492 (1985); Kunkel T. A. MethodsEnzymol. 85: 2763-2766 (1988)). Amino acids mutations may also occur innature. Thus, such a protein comprising an amino acid sequence in whichone or more amino acids in the sequence of the MC-PIR1 or MC-PIR2protein are mutated, and which is functionally equivalent to the MC-PIR1or MC-PIR2 protein, is also included in the present invention. In such amutant protein, the number of mutated amino acids is usually 50 or less,preferably 30 or less, and more preferably 10 or less (for example, fiveamino acids or less).

In mutating an amino acid , it is preferable to change it into anotheramino acid that allows the properties of the amino acid side chain to beconserved. Based on the properties of side chains, amino acids can bedivided into, for example, the following groups: hydrophobic amino acids(A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q,G, H, K, S, T), amino acids with an aliphatic side chain (G, A, V, L, I,P), amino acids with a side chain comprising a hydroxyl group (S, T, Y),amino acids with a side chain comprising sulfur (C, M), amino acids witha side chain comprising a carboxylic acid and an amide group (D, N, E,Q), basic amino acids (R, K, H), and aromatic amino acids (H, F, Y, W)(in the parentheses, amino acids are shown using the one letter code).

It is already known that a protein having a modified amino acidsequence, in which one or more amino acids are deleted, added, and/orsubstituted with another amino acid, can maintain the originalbiological activity (Mark D. F. et al. Proc. Natl. Acad. Sci. USA 81:5662-5666 (1984); Zoller M. J. and Smith M. Nucleic Acids Res. 10:6487-6500 (1982); Wang A. et al. Science 224: 1431-1433 (1984);Dalbadie-MeFarland G. et al. Proc. Natl. Acad. Sci. USA 79: 6409-6413(1982)).

A protein comprising an amino acid sequence in which multiple amino acidresidues are added to the sequence of MC-PIR1 or MC-PIR2 proteinincludes fusion proteins comprising these proteins. Fusion proteins suchas those between the proteins of this invention and other peptides orproteins are included in the present invention. To produce a fusionprotein, a DNA encoding the MC-PIR1 or MC-PIR2 protein (comprising theamino acid sequence according to SEQ ID NO: 2 or 4) and a DNA encodinganother peptide or protein are ligated so that their frames match, andintroduced into an expression vector to express in a host. Any methodcommonly known to those skilled in the art can be used. Any peptide orprotein may be used for making a fusion protein with a protein of thisinvention.

Known peptides that can be used as peptides that are fused to theproteins of the present invention include, for example, FLAG (Hopp, T.P. et al., Biotechnology (1988) 6, 1204-1210), 6×His containing sixhistidine (HIS) residues, 10×His, HA (Influenza agglutinin), human c-mycfragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag,SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment, and the like. Examples of proteins that may be fused toproteins of the invention include GST (glutathione-S-transferase), HA(Influenza agglutinin), immunoglobulin constant region, β-galactosidase,MBP (maltose-binding protein), and such.

Fusion proteins can be prepared by fusing commercially available DNAencoding the fusion peptides or proteins discussed above, with the DNAencoding the proteins of the present invention, and expressing theprepared fused DNA.

An alternative method known in the art to isolate functionallyequivalent proteins is, for example, the method using hybridization(Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold SpringHarbor Lab. Press, 1989). One skilled in the art can readily isolate aDNA having high homology with an entire or partial DNA sequence (SEQ IDNOs: 1 and 3) that encodes the MC-PIR1 and MC-PIR2 proteins, and isolateproteins functionally equivalent to the MC-PIR1 or MC-PIR2 protein usingthe isolated DNA.

The present invention includes proteins encoded by DNA that hybridizewith DNA encoding the MC-PIR1 or MC-PIR2 protein, and which arefunctionally equivalent to the MC-PIR1 or MC-PIR2 protein. Such proteinsinclude, for example, homologues in mice or other mammals (for example,a protein encoded by a human, rat, rabbit, or bovine homologous gene).

The conditions for hybridization used for isolating a DNA encoding aprotein functionally equivalent to the MC-PIR1 or MC-PIR2 protein can beappropriately selected by those skilled in the art. For example, lowstringent conditions may be used for hybridization. Low stringentconditions are post-hybridization washing in 0.1×SSC, 0.1% SDS at 42°C., for example, and preferably in 0.1×SSC, 0.1% SDS at 50° C. Highlystringent conditions are more preferable, which are washing in 5×SSC,0.1% SDS at 65° C., for example. Under these conditions, a DNA having ahigher homology can be efficiently obtained by increasing thetemperature. Multiple factors including the temperature, saltconcentration, and such are considered to affect the stringency ofhybridization; one skilled in the art can achieve similar stringenciesby appropriately selecting these factors.

In addition to hybridization, a gene amplification method such as thepolymerase chain reaction (PCR) may be used for gene isolation usingsynthesized primers based on the nucleotide sequence of the DNA encodingthe MC-PIR1 or MC-PIR2 protein (SEQ ID NO: 1 or 3).

Normally, such a protein encoded by the DNA isolated using the abovehybridization techniques or gene amplification, and which isfunctionally equivalent to the MC-PIR1 or MC-PIR2 protein, has a highhomology with these proteins (comprising the amino acid sequence of SEQID NO: 2 or 4) at the amino acid level. The proteins of this inventioninclude proteins functionally equivalent to the MC-PIR1 or MC-PIR2protein, and having a high homology with these proteins at the aminoacid level. High homology normally means an identity of at least 50% ormore at the amino acid level, preferably 75% or more, more preferably85% or more, and most preferably 95% or more (96% or more, 97% or more,98% or more, or 99% or more). Homology between proteins can bedetermined according to the algorithm described in literature (Wilbur W.J. and Lipman D. J. Proc. Natl. Acad. Sci. USA 80: 726-730 (1983)).

The proteins of the present invention may have variations in the aminoacid sequence, molecular weight, isoelectric point, or presence orcomposition of sugar chains, depending on the cell or host used forproducing it, or the method of purification, as described later on.Nevertheless, such proteins are included in the present invention aslong as they are functionally equivalent to the MC-PIR1 or MC-PIR2protein. For example, if a protein of the present invention is expressedin a prokaryotic cell such as E. coli, a methionine may be attached tothe N-terminus of the original protein. Such proteins are also includedin the present invention.

The proteins of the present invention can be prepared as recombinantproteins or natural proteins, by methods well known to those skilled inthe art. A recombinant protein can be prepared by: inserting a DNA thatencodes a protein of the present invention (for example, the DNAcomprising the nucleotide sequence of SEQ ID NO: 1 or 3), into anappropriate expression vector; introducing the vector into anappropriate host cell; collecting thus obtained recombinants; obtainingan extract thereof; and purifying the protein by subjecting the extractto a chromatography. Examples of chromatographies are ion exchangechromatography, reverse phase chromatography, gel filtration, oraffinity chromatography utilizing a column to which an antibody againsta protein of the present invention is immobilized, or combinations ofmore than one of the aforementioned columns.

When the protein of the present invention is expressed within host cells(for example, animal cells or E. coli) as a fusion protein with theglutathione-S-transferase protein, or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column. Afterpurifying the fusion protein, it is also possible to exclude regionsother than the objective protein by cutting with thrombin or factor-Xaas required.

A natural protein may be isolated by a method known to those skilled inthe art, for example, through purification by applying a tissue or cellextract expressing a protein of this invention onto an affinity columnin which an antibody (described below) capable of binding to the proteinhas been immobilized. Both monoclonal and polyclonal antibodies can beused.

In addition, the present invention comprises partial peptides of theproteins of this invention. A partial peptide of this inventioncomprises an amino acid sequence of at least seven residues or more,preferably eight residues or more, and more preferably nine residues ormore. The partial peptides may be useful, for example, for preparingantibodies against the proteins of this invention, in screenings forcompounds capable of binding to the proteins, and in screenings foractivators or inhibitors of the proteins. In addition, the peptides maybe useful by themselves as antagonists or competitors of the proteins ofthis invention. The partial peptides of this invention can be producedusing genetic engineering, by commonly known peptide synthesis methods,or digesting a protein of this invention with an appropriate peptidase.Peptide synthesis may be performed, for example, by either solid phasesynthesis or liquid phase synthesis.

A DNA encoding a protein of the present invention would be useful notonly for producing the protein in vivo or in vitro as described above,but also for applications in gene therapy of a disease caused by anabnormal function of the gene encoding the protein or a disease that canbe treated with the protein, DNA diagnostics, etc. A DNA of thisinvention can take any form as long as it encodes a protein of thisinvention. It can be a cDNA synthesized from mRNA, genomic DNA, orchemically-synthesized DNA. In addition, it includes a DNA comprisingany nucleotide sequence based on the degeneracy of genetic code as longas it encodes a protein of this invention.

A DNA of this invention can be prepared by methods commonly known tothose skilled in the art. For example, it may be prepared by making acDNA library from cells expressing a protein of this invention, andperforming hybridization using a partial nucleotide sequence of the DNA(for example, SEQ ID NO: 1 or 3) as a probe. The cDNA library may beprepared, for example, according to the method described in literature(Sambrook J. et al. Molecular Cloning, Cold Spring Harbor LaboratoryPress (1989)), or obtained from a commercial source. Alternatively, theDNA of this invention may be prepared as follows: RNA is prepared fromcells expressing a protein of this invention, from which cDNA issynthesized using reverse transcriptase. Then, oligo DNA is synthesizedbased on the sequence of the DNA (for example, SEQ ID NO: 1 or 3), andused as a primer in a PCR reaction to amplify a cDNA encoding a proteinof this invention.

Furthermore, the coding region of the cDNA can be determined bydetermining the nucleotide sequence of the obtained cDNA, and the aminoacid sequence of a protein of this invention can be thus obtained. Inaddition, the obtained cDNA may be used as a probe for screening agenomic DNA library to isolate a genomic DNA.

Specific procedures are as follows: First, mRNA is isolated from a cell,tissue, or organ expressing a protein of this invention (for example,mast cells or tissues in which expression was detected by RT-PCR in theExample below). mRNA may be isolated by preparing total RNA using acommonly known method such as guanidine ultracentrifugation (Chirgwin J.M. et al. Biochemistry 18: 5294-5299 (1979)), or AGPC method(Chomczynski P. and Sacchi N. Anal. Biochem. 162: 156-159 (1987)), andthen purifying mRNA from total RNA using an mRNA Purification Kit(Pharmacia), etc. Alternatively, mRNA may be directly prepared using theQuickPrep mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized by using a commercially availablekit such as the AMV Reverse Transcriptase First-strand cDNA SynthesisKit (Seikagaku Kogyo). Alternatively, using a primer described herein,or such, cDNA may be synthesized and amplified following the 5′-RACEmethod (Frohman M. A. et al. Proc. Natl. Acad. Sci. U.S.A. 85:8998-9002(1988); Belyavsky A. et al. Nucleic Acids Res. 17:2919-2932 (1989))using the 5′-Ampli FINDER RACE Kit (Clontech) and the polymerase chainreaction (PCR).

A desired DNA fragment is prepared from PCR products and ligated with avector DNA to produce a recombinant vector construct. This construct isused to transform E. coli or such, and desired recombinant vectors areprepared from a selected colony/colonies. The nucleotide sequence of thedesired DNA can be verified by conventional methods such asdideoxynucleotide chain termination.

The nucleotide sequence of a DNA of the invention may be designed to beexpressed more efficiently by taking into account the frequency of codonusage in the host used for the expression (Grantham R. et al. NucleicAcids Res. 9:43-74 (1981)). The DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of an initiation codon (ATG) and/or astop codon (TAA, TGA, or TAG).

The DNA of this invention specifically includes a DNA comprising thenucleotide sequence starting from “a” at 148 through “g” at 1101 in thenucleotide sequence of SEQ ID NO: 1, or a DNA comprising the nucleotidesequence from “a” at 1 through “g” at 684 in the sequence of SEQ ID NO:3.

In addition, the DNA of this invention includes a DNA that hybridizeswith a DNA comprising the nucleotide sequence shown as SEQ ID NO: 1 or 3and encodes a protein functionally equivalent to an above-describedprotein of this invention. Hybridization conditions may be appropriatelychosen by one skilled in the art. Specifically, the above-describedspecific conditions may be used. Under these conditions, the higher thetemperature, the higher the homology of the obtained DNA would be. Theabove hybridizing DNA is preferably a naturally-occurring DNA, forexample, a cDNA or chromosomal DNA.

The present invention also provides a vector into which a DNA of thepresent invention has been inserted. A vector of the present inventionis useful to maintain a DNA of the present invention in a host cell, orto express a protein of the present invention.

When E. coli is a host cell and the vector is amplified and produced ina large amount in E. coli (e.g., JM109, DH5α, HB101, or XL1Blue), thevector should have “ori” to be amplified in E. coli and a marker genefor selecting transformed E. coli (e.g., a drug-resistance gene selectedby a drug such as ampicillin, tetracycline, kanamycin, chloramphenicol,or the like). For example, M13-series vectors, pUC-series vectors,pBR322, pBluescript, pCR-Script, etc. can be used. In addition, pGEM-T,pDIRECT, and pT7 can also be used for subcloning and extracting cDNA aswell as the vectors described above. When a vector is used to produce aprotein of the present invention, an expression vector is especiallyuseful. For example, an expression vector to be expressed in E. colishould have the above characteristics to be amplified in E. coli. When Ecoli such as JM109, DH5α, HB101, or XL1 Blue are used as a host cell,the vector should have a promoter, for example, the lacZ promoter (Wardet al., Nature (1989) 341, 544-546; FASEB J (1992) 6, 2422-2427), araBpromoter (Better et al., Science (1988) 240, 1041-1043), or T7 promoteror the like, that can efficiently express the desired gene in E. coli.In this respect, pGEX-5X-1 (Pharmacia), “QIAexpress system” (Qiagen),pEGFP and pET (in this case, the host is preferably BL21 which expressesT7 RNA polymerase), for example, can be used in addition to the abovevectors.

Additionally, the vector may also contain a signal sequence forpolypeptide secretion. An example of a signal sequence that directs theprotein to be secreted to the periplasm of the E. coli is the pelBsignal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379). Meansfor introducing the vectors into target host cells include, for example,the calcium chloride method and the electroporation method.

In addition to E. coli, for example, expression vectors derived frommammals (for example, pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids.Res. 1990, 18 (17), p5322), pEF, pCDM8), expression vectors derived frominsect cells (for example, “Bac-to-BAC baculovirus expression system”(GIBCO BRL), pBacPAK8), expression vectors derived from plants (forexample pMH1, pMH2), expression vectors derived from animal viruses (forexample, pHSV, pMV, pAdexLcw), expression vectors derived fromretroviruses (for example, pZIPneo), expression vector derived fromyeast (for example, “Pichia Expression Kit” (Invitrogen), pNV11,SP-Q01), and expression vectors derived from Bacillus subtilis (forexample, pPL608, pKTH50) can be used as vectors for producing a proteinof the present invention.

In order to express the vector in animal cells such as CHO, COS, orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al., Nature(1979) 277, 108), the MMLV-LTR promoter, the EF1α promoter (Mizushima etal., Nucleic Acids Res. (1990) 18, 5322), the CMV promoter, and thelike, and preferably a marker gene for selecting transformants (forexample, a drug resistance gene selected by a drug (e.g., neomycin,G418)). Examples of known vectors with these characteristics include,for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

In addition, when the aim is to stably express a gene and at the sametime increase the copy number of the gene in cells, one can use themethod of introducing, into CHO cells in which the nucleic acidsynthesizing pathway is deleted, a vector comprising the complementaryDHFR gene (for example pCHO I) and then amplifying this by methotrexate(MTX). Furthermore, when the aim is to transiently express a gene, onecan use the method of transfecting a vector comprising a replicationorigin of SV40 (pcD, etc.) into COS cells comprising the SV40 T antigenexpressing gene on the chromosome. The replication origin may also bederived from the polyoma virus, adenovirus, bovine papilloma virus(BPV), and the like. Furthermore, the expression vector may carry, as aselection marker, the aminoglycoside transferase (APH) gene, thymidinekinase (TK) gene, E. coli xanthine-guanine-phosphoribosyl transferase(Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and such, forincreasing the copy number in the host cell system.

A DNA of the present invention can further be expressed in vivo inanimals, for example, by inserting the DNA into an appropriate vectorand introducing it into living bodies by methods such as the retrovirusmethod, the liposome method, the cationic liposome method, and theadenovirus method. Gene therapy against diseases attributed to mutationof a gene encoding a protein of the present invention can be thusaccomplished. An adenovirus vector (for example pAdexlcw) or retrovirusvector (for example, pZIPneo) can be given as an example of a vector,but the vector is not restricted thereto. General gene manipulations,such as insertion of a DNA of the present invention to a vector, can beperformed according to conventional methods (Molecular Cloning,5.61-5.63). Administration into a living body can be either an ex vivomethod, or in vivo method.

The present invention further provides a host cell into which a vectorof the present invention has been transfected. The host cell into whicha vector of the invention is transfected is not particularly limited.For example, E. coli, various animal cells and such can be used. Thehost cells of the present invention can be used, for example, as aproduction system for producing or expressing a protein of the presentinvention. The present invention provides methods of producing a proteinof the invention both in vitro and in vivo. For in vitro production,eukaryotic cells or prokaryotic cells can be used as host cells.

Useful eukaryotic cells may be animal, plant, or fungi cells. Animalcells include, for example, mammalian cells such as CHO (J. Exp. Med.108:945 (1995)), COS, 3T3, myeloma, baby hamster kidney (BHK), HeLa, orVero cells; amphibian cells such as Xenopus oocytes (Valle et al. Nature291:340-358 (1981)); or insect cells such as Sf9, Sf21, or Tn5 cells.CHO cells lacking the DHFR gene (dhfr-CHO) (Proc. Natl. Acad. Sci.U.S.A. 77:4216-4220 (1980)) or CHO K-1 (Proc. Natl. Acad. Sci. U.S.A.60:1275 (1968)) may also be used. Of animal cells, CHO cells areparticularly preferable for mass expression. A vector can be transfectedinto host cells by, for example, the calcium phosphate method, theDEAE-dextran method, the cationic liposome DOTAP (Boehringer Mannheim),the electroporation method, the lipofection method, and so on.

As plant cells, plant cells derived from Nicotiana tabacum are known asprotein-production systems, and may be used as callus cultures. As fungicells, yeast cells such as Saccharomyces, including Saccharomycescerevisiae, or filamentous fungi such as Aspergillus, includingAspergillus niger, are known and may be used herein.

Useful prokaryotic cells include bacterial cells such as E. coli, forexample, JM109, DH5α, and HB101. Other bacterial systems includeBacillus subtilis.

These cells are transformed by a desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. Culture medium for animal cellsinclude, for example, DMEM, MEM, RPMI 1640, and IMDM. These may be usedwith or without a serum supplement such as the fetal calf serum (FCS).The pH of the culture medium is preferably between about 6 and 8. Suchcells are typically cultured at about 30 to 40° C. for about 15 to 200hr, and the culture medium may be replaced, aerated, or stirred ifnecessary.

Animal or plant hosts may be used for the in vivo production. Forexample, a desired DNA can be transfected into an animal or plant host.Encoded proteins are produced in vivo, and then recovered. These animaland plant hosts are included in the host cells of the present invention.

Animals used for the production system described above include, but arenot limited to, mammals and insects. Mammals, such as goats, pigs,sheep, mice and cows, may be used (Vicki Glaser, SPECTRUM BiotechnologyApplications (1993)). Alternatively, the mammals may be transgenicanimals.

For instance, a desired DNA may be prepared as a fusion gene, by fusingit with a gene such as the goat β casein gene which encodes a proteinspecifically produced into milk. DNA fragments comprising the fusiongene are injected into goat embryos, which are then implanted in femalegoats. Proteins are recovered from milk produced by the transgenic goats(i.e., those born from the goats that had received the embryos) or fromtheir offspring. To increase the amount of milk containing the proteinsproduced by the transgenic goats, appropriate hormones may beadministered to them (Ebert K. M. et al. Bio/Technology 12:699-702(1994)).

Alternatively, insects, such as the silkworm, may be used. A DNAencoding a desired protein inserted into baculovirus can be used totransfect silkworms, and the desired protein may be recovered from theirbody fluid (Susumu M. et al. Nature 315: 592-594 (1985)).

As plants, for example, tobacco can be used. When using tobacco, a DNAencoding a desired protein may be inserted into a plant expressionvector such as pMON530, which is introduced into bacteria such asAgrobacterium tumefaciens. Then, the bacteria are used to transfect atobacco plant such as Nicotiana tabacum, and a desired polypeptide isrecovered from the leaves (Julian K. -C. Ma et al., Eur. J. Immunol. 24:131-138 (1994)).

A protein of the present invention obtained as above may be isolatedfrom the inside or outside (such as culture medium) of host cells, andpurified as a substantially pure homogeneous protein. The method forprotein isolation and purification is not limited to any specificmethod, and any standard method may be used. For instance, columnchromatography, filter, ultrafiltration, salt precipitation, solventprecipitation, solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis, dialysis, and recrystallization may be appropriatelyselected and combined to isolate and purify the protein.

Examples of chromatographies include, for example, affinitychromatography, ion-exchange chromatography, hydrophobic chromatography,gel filtration, reverse phase chromatography, adsorption chromatography,and such (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed. Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). These chromatographies may be performedby a liquid chromatography such as HPLC and FPLC. Thus, the presentinvention provides highly purified proteins prepared by the abovemethods.

A protein of the present invention may be optionally modified orpartially deleted by treating it with an appropriate proteinmodification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, glucosidase and so on.

The present invention provides antibodies that bind to the proteins ofthe invention. The antibodies can take any form such as monoclonal orpolyclonal antibodies, and includes antiserum obtained by immunizing ananimal such as a rabbit with a protein of the invention, all classes ofpolyclonal and monoclonal antibodies, human antibodies, and humanizedantibodies produced by genetic recombination.

A protein of the invention used as an antigen to obtain an antibody maybe derived from any animal species, but is preferably derived from amammal such as a human, mouse, or rat, more preferably a human. Ahuman-derived protein may be obtained from the nucleotide or amino acidsequences disclosed herein.

According to the present invention, the protein to be used as animmunization antigen may be a complete protein or a partial peptide ofthe protein. A partial peptide may comprise, for example, the amino(N)-terminal or carboxy (C)-terminal fragment of a protein of thepresent invention. Herein, an antibody is defined as a protein thatreacts with either the whole protein of the present invention, or afragment of the protein.

A gene encoding a protein of the invention or its fragment may beinserted into a known expression vector, which is then used to transforma host cell as described herein. The desired protein or its fragment maybe recovered from the outside or inside of host cells by any standardmethod, and may subsequently be used as an antigen. Alternatively, cellsexpressing the protein or their lysates, or a chemically synthesizedprotein may be used as the antigen. In the case of a short peptide, itis preferably bound to an appropriate carrier protein such as keyholelimpet hemocyanin, bovine serum albumin, and ovalbumin before using asantigen.

Any mammalian animal may be immunized with the antigen, but preferably,the compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha, or Primates areused.

Animals of Rodentia include, for example, mice, rats, and hamsters.Animals of Lagomorpha include, for example, rabbits. Animals of Primatesinclude, for example, monkeys of Catarrhini (old world monkeys) such asMacaca fascicularis, rhesus monkeys, sacred baboons, and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method of immunization for mammals. More specifically, antigensmay be diluted and suspended in an appropriate amount of phosphatebuffered saline (PBS), physiological saline, etc. If desired, theantigen suspension may be mixed with an appropriate amount of a standardadjuvant such as Freund's complete adjuvant, made into an emulsion, andthen administered to mammalian animals. Preferably, this is followed byseveral administrations of antigen mixed with an appropriately amount ofFreund's incomplete adjuvant every 4 to 21 days. An appropriate carriermay also be used for immunization. After immunizing as above, serum isexamined by a standard method for an increase in the amount of desiredantibodies.

Polyclonal antibodies against the proteins of the present invention maybe prepared by collecting blood from the immunized mammal afterverifying an increase of desired antibodies in the serum, and byseparating serum from the blood by any conventional method. Polyclonalantibodies include serum containing polyclonal antibodies, as well asfractions containing the polyclonal antibodies isolated from the serum.Immunoglobulin G or M can be prepared from a fraction which recognizesonly the protein of the present invention using, for example, anaffinity column coupled with a protein of the present invention, andfurther purifying this fraction using a protein A or protein G column.

To prepare monoclonal antibodies, immunocytes are collected from themammal immunized with the antigen and checked for an increase in thelevel of desired antibodies in the serum as described above, and aresubjected to cell fusion. The immune cells used for cell fusion arepreferably obtained from the spleen. Other preferred parental cells tobe fused with the above immunocytes include, for example, mammalianmyeloma cells, and more preferably myeloma cells having an acquiredproperty for the selection of fused cells by drugs.

The above immunocytes and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al. (Galfre, Q andMilstein, C., Methods Enzymol. (1981) 73, 346).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium such as the HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, which is sufficient to allow all the other cells, withthe exception of the desired hybridoma (non-fused cells), to die. Then,standard limiting dilution is performed to screen and clone a hybridomaproducing the desired antibody.

In addition to the above method in which a non-human animal is immunizedwith an antigen for preparing a hybridoma, a hybridoma producing adesired human antibody that is able to bind to a protein can be obtainedby the following method. First, human lymphocytes such as those infectedby the EB virus may be immunized with a protein, protein expressingcells, or their lysates in vitro. Then, the immunized lymphocytes arefused with human-derived myeloma cells that are capable of indefinitedivision, such as U266, to yield the desired hybridoma (UnexaminedPublished Japanese Patent Application No. (JP-A) Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are harvested. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which a protein of the presentinvention is coupled. The antibodies of the present invention can beused not only for purification and detection of the proteins of thepresent invention, but also as candidates for agonists and antagonistsof the proteins. In addition, these antibodies can be applied to theantibody treatment for diseases related to the proteins of the presentinvention. When an obtained antibody is to be administered to the humanbody (antibody treatment), a human antibody or a humanized antibody ispreferable to reduce immunogenicity.

For example, transgenic animals having a repertoire of human antibodygenes may be immunized with an antigen selected from a protein, cellsexpressing the protein or their lysates. Antibody producing cells arethen collected from the animals and fused with myeloma cells to obtainhybridomas, from which human antibodies against the protein can beprepared (see WO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735,and WO96-34096).

Alternatively, an immunocyte that produces antibodies, such as animmunized lymphocyte, may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can also be recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck C. A.K. and Larrick J. W. Therapeutic Monoclonal Antibodies, published in theUnited Kingdom by MacMillan Publishers LTD (1990)). A DNA encoding anantibody may be cloned from an immunocyte, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. The present invention also provides recombinantantibodies prepared as described above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, so long as it binds to one or more ofthe proteins of the invention. For instance, the antibody fragment maybe Fab, F(ab′)₂, Fv, or single chain Fv (scFv), in which Fv fragmentsfrom H and L chains are ligated by an appropriate linker (Huston J. S.et al. Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988)). Morespecifically, an antibody fragment may be generated by treating anantibody with an enzyme such as papain or pepsin. Alternatively, a geneencoding the antibody fragment may be constructed, inserted into anexpression vector, and expressed in an appropriate host cell (see, forexample, Co M. S. et al. J. Immunol. 152:2968-2976 (1994); Better M. andHorwitz A. H. Methods Enzymol. 178:476-496 (1989); Pluckthun A. andSkerra A. Methods Enzymol. 178:497-515 (1989); Lamoyi E. MethodsEnzymol. 121:652-663 (1986); Rousseaux J. et al. Methods Enzymol.121:663-669 (1986); Bird R. E. and Walker B. W. Trends Biotechnol.9:132-137 (1991)).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides suchmodified antibodies. The modified antibody can be obtained by chemicallymodifying an antibody. These modification methods are conventional inthe field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody between a variable region derived from a nonhumanantibody and a constant region derived from a human antibody. It canalso be obtained as a humanized antibody comprising acomplementarity-determining region (CDR) derived from a nonhumanantibody, a frame work region (FR) and a constant region derived from ahuman antibody. Such antibodies can be prepared using a knowntechnology.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by appropriately selecting and combining columnchromatographies such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing, and others (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory, 1988), but the chromatographies are not limited thereto. Theconcentration of the thus obtained antibodies can be determined bymeasuring the absorbance, by an enzyme-linked immunosorbent assay(ELISA), and so on.

A protein A column or protein G column can be used as the affinitycolumn. Examples of protein A columns include, for example, Hyper D,POROS, and Sepharose F.F. (Pharmacia).

Examples of chromatographies other than affinity chromatographyincludes, for example, ion-exchange chromatography, hydrophobicchromatography, gel filtration, reverse-phase chromatography, adsorptionchromatography, and the like (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press, 1996). The chromatographiescan be carried out by a liquid-phase chromatography, such as HPLC, FPLC.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/orimmunofluorescence may be used to measure the antigen binding activityof an antibody of the invention. In ELISA, an antibody of the presentinvention is immobilized on a plate, a protein of the invention isapplied to the plate, and then a sample containing a desired antibody,such as a culture supernatant of antibody producing cells or purifiedantibodies, is applied. Then, a secondary antibody that recognizes theprimary antibody and is labeled with an enzyme such as alkalinephosphatase is applied, and the plate is incubated. Next, after washing,an enzyme substrate such as p-nitrophenyl phosphate is added to theplate, and the absorbance is measured to evaluate the antigen bindingactivity of the sample. A fragment of the protein, such as a C-terminalfragment may be used as the protein. BIAcore (Pharmacia) may be used toevaluate the activity of an antibody according to the present invention.

These methods allow the detection or measurement of a protein of theinvention by exposing an antibody of the invention to a sample assumedto contain the protein of the invention, and detecting or measuring theimmune complex formed by the antibody and the protein. Because themethod of detection or measurement of the protein according to theinvention can specifically detect or measure a protein, the method maybe useful in a variety of experiments in which the protein is used.

Furthermore, the present invention provides a polynucleotide comprisingat least 15 nucleotides, which is complementary to a DNA encoding theMC-PIR1 or MC-PIR2 protein (comprising the nucleotide sequence accordingto SEQ ID NO: 1 or 3) or a complementary strand thereof.

Herein, “complementary strand” means a strand that is opposite relativeto the other strand in a double-stranded nucleic acid composed of A:T (Uin the case of RNA) and G:C base pairs. In addition, being“complementary” is not limited to having completely complementarity in acontinuous region of at least 15 nucleotides, but it can also meanhaving a homology of at least 70%, preferably at least 80%, morepreferably 90%, and most preferably 95% or higher at the nucleotidelevel. Homology can be determined using the algorithm described herein.

Such nucleic acids include: probes or primers used for detecting oramplifying a DNA encoding a protein of this invention; probes or primersused for detecting DNA expression; or nucleotides or nucleotidederivatives (for example, antisense oligonucleotides or ribozymes, orDNA encoding them) used for regulating the expression of a protein ofthis invention. Such nucleic acids may also be useful for preparing DNAchips.

When using as a primer, the 3′-region can be made complementary and arecognition site for a restriction enzyme, or a tag can be attached tothe 5′-region.

Antisense oligonucleotides include, for example, those that hybridizewith any site in the nucleotide sequence of SEQ ID NO: 1 or 3. Suchantisense oligonucleotides are preferably complementary to at least 15or more continuous nucleotides in the sequence of SEQ ID NO: 1 or 3.More preferably, they are complementary to at least 15 or morecontinuous nucleotides comprising the translation initiation codon inthe sequence.

Derivatives or modified forms of antisense oligonucleotides can be usedas antisense oligonucleotides. For example, modified antisenseoligonucleotides include lower alkylphosphonate modified forms such asthe methylphosphonate type or ethylphosphonate type, or phosphorothioatemodified forms, phosphoroamidate modified forms, or the like.

Antisense oligonucleotides are not limited to those in which the entirenucleotide sequence corresponding to a nucleotide sequence composing acertain DNA or mRNA region is completely complementary. One or morenucleotide mismatches may be contained as long as the antisenseoligonucleotide specifically hybridizes with the nucleotide sequenceshown in SEQ ID NO: 1 or 3.

The antisense oligonucleotide derivatives or modified forms of thepresent invention act upon cells producing a protein of the invention bybinding to the DNA or mRNA encoding the protein, inhibiting itstranscription or translation, promoting the degradation of the mRNA, andinhibiting the expression of the protein, thereby resulting in theinhibition of the protein's function.

An antisense oligonucleotide derivative or modified form of the presentinvention can be made into an external preparation, such as a linimentor a poultice, by mixing with a suitable base material which is inactiveagainst the derivative or modified form.

Also, as needed, the derivatives or modified forms can be formulatedinto tablets, powders, granules, capsules, liposome capsules,injections, solutions, nose-drops, and freeze-drying agents by addingexcipients, isotonic agents, solubilizers, stabilizers, preservatives,pain-killers, and such. These can be prepared by following usualmethods.

The antisense oligonucleotide derivatives or modified forms are given toa patient by directly applying them onto the ailing site or by injectingthem into a blood vessel so that they will reach the site of ailment. Anantisense-mounting medium can also be used to increase durability andmembrane-permeability. Examples are, liposomes, poly-L-lysine, lipids,cholesterol, lipofectin or derivatives of these.

The dosage of an antisense oligonucleotide derivative or modified formof the present invention can be suitably adjusted according to thepatient's condition and used in desired amounts. For example, a doserange of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can beadministered.

The antisense oligonucleotides of the invention inhibits the expressionof a protein of the invention and is thereby useful for suppressing thebiological activity of the protein. Also, expression-inhibitorscomprising an antisense oligonucleotide of the invention are useful inthat they can inhibit the biological activity of a protein of theinvention.

The proteins of this invention may be useful for screening compoundscapable of binding to the proteins. Specifically, the proteins can beused in methods of screening for compounds capable of binding to theproteins, comprising the steps of contacting a protein of the inventionand a test sample that is expected to contain such a compound, and thenselecting the compound.

The protein of the invention used for the screening may be a recombinantprotein or naturally-occurring protein. It may also be a partialpeptide. It can be expressed on the cell surface, or contained in themembrane fraction. The test sample is not limited to any particularsample; it can be, for example, a cell extract, a cell culturesupernatant, a product of a fermentation microorganism, a marineorganism extract, a plant extract, a purified or crude protein, apeptide, a non-peptide compound, a synthetic low molecular weightcompound, or a natural compound. The protein of this invention can becontacted with the test sample as a purified protein, soluble protein,in a form bound to a carrier, as a fusion protein with another protein,in a form expressed on the cell surface, or as a form contained in themembrane fraction.

As a method of screening for proteins that, for example, bind to aprotein of the present invention using a protein of the presentinvention, many methods well known to those skilled in the art can beused. Such a screening can be conducted by, for example, theimmunoprecipitation method, specifically, in the following manner. Agene encoding a protein of the present invention is expressed in animalcells, or such, by inserting the gene into an expression vector forforeign genes, such as pSV2neo, pcDNA I, and pCD8. The promoter to beused for the expression may be any promoter that can generally be usedand include, for example, the SV40 early promoter (Rigby in Williamson(ed.), Genetic Engineering, vol. 3. Academic Press, London, p. 83-141(1982)), the EF-1α promoter (Kim et al., Gene 91, p217-223 (1990)), theCAG promoter (Niwa et al. Gene 108, p. 193-200 (1991)), the RSV LTRpromoter (Cullen Methods in Enzymology 152, p. 684-704 (1987)) the SRαpromoter (Takebe et al., Mol. Cell. Biol. 8, p. 466 (1988), the CMVimmediate early promoter (Seed and Aruffo Proc. Natl. Acad. Sci. USA 84,p. 3365-3369 (1987)), the SV40 late promoter (Gheysen and Fiers J. Mol.Appl. Genet. 1, p. 385-394 (1982)), the Adenovirus late promoter(Kaufman et al., Mol. Cell. Biol. 9, p. 946 (1989)), the HSV TK promoterand so on.

The introduction of the gene into animal cells to express a foreign genecan be performed according to any method, for example, theelectroporation method (Chu G et al. Nucl. Acids Res. 15, 1311-1326(1987)), the calcium phosphate method (Chen, C and Okayama, H. Mol.Cell. Biol. 7, 2745-2752 (1987)), the DEAE dextran method (Lopata, M. A.et al. Nucl. Acids Res. 12, 5707-5717 (1984)), Sussman, D. J. andMilman, G. Mol. Cell. Biol. 4, 1642-1643 (1985)), the Lipofectin method(Derijard, B. Cell 7, 1025-1037 (1994); Lamb, B. T. et al. NatureGenetics 5, 22-30 (1993): Rabindran, S. K. et al. Science 259, 230-234(1993)), and so on.

A protein of the present invention can be expressed as a fusion proteincomprising a recognition site (epitope) of a monoclonal antibody byintroducing, to the N- or C-terminus of the protein, an epitope of amonoclonal antibody whose specificity has been revealed. A commerciallyavailable epitope-antibody system can be used (Experimental Medicine 13,85-90 (1995)). Vectors that can express a fusion protein with, forexample, p-galactosidase, maltose-binding protein, glutathioneS-transferase, green florescence protein (GFP) and so on throughmultiple cloning sites are commercially available.

A method of preparing a fusion protein by introducing only a smallepitope portion consisting of several to a dozen amino acids so as tonot change, as much as possible, the property of the protein of thepresent invention by the fusion, has also been reported. Epitopes, suchas polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG,Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein(T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (anepitope on monoclonal phage), and such, and monoclonal antibodiesrecognizing them can be used as epitope-antibody systems for screeningproteins binding to the proteins of the present invention (ExperimentalMedicine 13, 85-90 (1995)).

In immunoprecipitation, an immune complex is formed by adding theseantibodies to a cell lysate prepared by using an appropriate detergent.The immune complex consists of a protein of the present invention, aprotein that can bind with the protein, and an antibody.Immunoprecipitation can also be conducted by using antibodies against aprotein of the present invention, besides using antibodies against theabove epitopes. An antibody against a protein of the present inventioncan be prepared, for example, by introducing a gene encoding the proteininto an appropriate E. coli expression vector, expressing the gene in E.coli, purifying the expressed protein, and immunizing rabbits, mice,rats, goats, chicken and such with the protein. The antibody can be alsoprepared by immunizing an animal of above with a synthesized partialpeptide of the protein of the present invention.

An immune complex can be precipitated, for example by Protein ASepharose or Protein G sepharose when the antibody is a mouse IgGantibody. If the proteins of the present invention are prepared asfusion proteins with an epitope such as GST, an immune complex can beformed in the same manner as when using the antibody against a proteinof the present invention, by using a substance that specifically bindsto the epitope, such as glutathione-Sepharose 4B.

Immunoprecipitation can be performed by following or according to, forexample, methods in literature (Harlow, E. and Lane, D.: Antibodies pp.511-552, Cold Spring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analyzing immunoprecipitated proteins. Thebound protein can be analyzed by the molecular weight of the proteinusing a gel with an appropriate concentration. Since the protein boundto the protein of the present invention is difficult to detect by acommon staining method such as Coomassie staining or silver staining,the detection sensitivity of the protein can be improved by culturingcells in a culture medium containing the radioactive isotope³⁵S-methionine or ³⁵S-cystein, labeling proteins within the cells, anddetecting the proteins. Once the molecular weight of the protein hasbeen revealed, the target protein can be purified directly from theSDS-polyacrylamide gel and its sequence can be determined.

In addition, as a method for isolating a protein capable of binding tothe protein using a protein of this invention, western blotting may beused (Skolnik E. Y. et al. Cell 65: 83-90 (1991)). Specifically, a cDNAlibrary using a phage vector (λgt11, ZAP, and the like) can be preparedusing a cell, tissue, or organ expected to express a protein capable ofbinding to the protein of this invention (for example, adipocytes ortissues where the expression is detected by northern blotting in theExamples). Then, the cDNA library can be expressed on LB-agarose,expressed protein immobilized onto a filter, the protein of thisinvention, which is purified and labeled, incubated with the abovefilter, and the plaque expressing the protein capable of binding to theprotein of this invention detected by the label. For labeling theprotein of this invention, methods that make use of: the binding betweenbiotin and avidin; an antibody specifically binding to the protein ofthis invention, or a peptide or polypeptide fused to the protein (forexample, GST); radioisotopes; or fluorescence, or the like, can be used.

In another embodiment of the screening methods of this invention, thetwo-hybrid system using cells may be used (Fields S. and Sternglanz R.Trends Genet. 10: 286-292 (1994); Dalton S. and Treisman R.Characterization of SAP-1, a protein recruited by serum response factorto the c-fos serum response element. Cell 68: 597-612 (1992);“MATCHMAKER Two-Hybrid System”; “Mammalian MATCHMAKER Two-Hybrid AssayKit”; “MATCHMAKER One-Hybrid System” (all from Clontech); and “HybriZAPTwo-Hybrid Vector System” (Stratagene)). In the two-hybrid system, aprotein of this invention or a partial peptide may be expressed in yeastcells as a fusion protein with the SRF DNA binding domain, or GAL4 DNAbinding domain. A cDNA library in which the protein is expressed as afusion between the VP16 or GAL4 transcription activation domain isprepared from cells in which a protein capable of binding to a proteinof this invention is expected to be present. The library is transfectedinto yeast cells, and cDNA derived from the library is isolated from apositive clone detected (when a protein capable of binding to theprotein of this invention is expressed in yeast cells, binding of thetwo proteins activates a reporter gene, which is used to detect apositive clone). Isolated cDNA may be introduced and expressed in E.coli to obtain a protein encoded by the cDNA. The reporter gene used inthe two-hybrid system may be, for example, a gene such as HIS3, Ade2,LacZ, CAT, luciferase, and PAI-1 (plasminogen activator inhibitor typeI), but is not limited thereto. Such a screening using the two-hybridsystem may be performed using a mammalian cell other than yeast.

A compound binding to a protein of the present invention can be screenedusing affinity chromatography. For example, the protein of the inventionmay be immobilized on a carrier of an affinity column, and a test samplepresumed to express a protein capable of binding to the protein of theinvention, is applied to the column. Herein, a test sample may be, forexample, a cell extract, cell lysate, etc. After loading the testsample, the column is washed, and proteins bound to the protein of theinvention can be prepared.

The amino acid sequence of the obtained protein is analyzed, an oligoDNA is synthesized based on the sequence, and cDNA libraries arescreened using the oligo DNA as a probe to obtain a DNA encoding theprotein.

A biosensor using the surface plasmon resonance phenomenon may be usedas a means for detecting or quantifying the bound compound in thepresent invention. When such a biosensor is used, the interactionbetween the protein of the invention and a test compound can be observedreal-time as a surface plasmon resonance signal, using only a minuteamount of protein and without labeling (for example, BIAcore,Pharmacia). Therefore, it is possible to evaluate the binding betweenthe protein of the invention and a test compound using a biosensor suchas BIAcore.

The methods of screening for molecules that bind when the immobilizedprotein of the present invention is exposed to synthetic chemicalcompounds, or natural substance banks, or a random phage peptide displaylibrary, or the methods of screening using high-throughput based oncombinatorial chemistry techniques (Wrighton Nc, Farrel F X, Chang R,Kashyap A K, Barbone F P, Mulcahy L S, Johnson D L, Barret R W, JolliffeL K, Dower W J; Small peptides as potent mimetics of the protein hormoneerythropoietin, Science (UNITED STATES) Jul. 26, 1996, 273 p 458-64,Verdine G L., The combinatorial chemistry of nature. Nature (ENGLAND)Nov. 7, 1996, 384, p 11-13, Hogan J C Jr., Directed combinatorialchemistry. Nature (ENGLAND) Nov. 7, 1996, 384 p17-9) to isolate not onlyproteins but chemical compounds that bind to a protein of the presentinvention (including agonists and antagonists) are well known to thoseskilled in the art.

In addition, the present inventors demonstrated that the proteins ofthis invention are capable of binding to SHP-1, SHP-2, SHIP, DAP10,DAP12, or FcRγ protein. Thus, using the above describedimmunoprecipitation or the two-hybrid system, the binding abilitybetween a protein of this invention and SHP-1, SHP-2, SHIP, DAP10,DAP12, or FcRγ protein in the presence of a test sample can be detected,and a substance with the ability to reduce the binding may be selectedto screen for a candidate for a medicinal compound. Thus, the presentinvention provides a screening method comprising the steps of contactinga protein of this invention with a protein selected from the groupconsisting of SHP-1, SHP-2, SHIP, DAP 10, DAP12, and FcRγ proteins inthe presence of a test sample, detecting the binding activity, andselecting a substance capable of reducing the binding activity bycomparing with the activity detected in the absence of the test sample.

Such compounds isolated using the screening methods of this inventioncan be candidates of therapeutic agents for regulating the activity of aprotein of this invention. They may be useful in applications such astreatment of diseases caused by abnormal function or abnormal expressionof the protein, or diseases that can be treated by regulating theactivity of the protein. Such diseases include allergic diseases such asatopic dermatitis, rhinitis, and asthma. Also included in the compoundscapable of binding to proteins of this invention are substances in whicha part of the structure of a compound isolated using a screening methodis modified by addition, deletion, and/or substitution.

When administrating a protein of this invention or a compound isolatedby a screening method of the invention as a pharmaceutical for humansand other mammals such as mice, rats, guinea-pigs, rabbits, chicken,cats, dogs, sheep, pigs, cattle, monkeys, baboons and chimpanzees, theprotein or the isolated compound can be directly administered orformulated into a dosage form using known pharmaceutical preparationmethods. For example, according to the need, the pharmaceutical can betaken orally, as a sugar-coated tablet, capsule, elixir or microcapsule,or non-orally, in the form of an injection of a sterile solution orsuspension with water or any other pharmaceutically acceptable liquid.For example, the compounds can be mixed with pharmacologicallyacceptable carriers or medium, specifically, sterilized water,physiological saline, plant oils, emulsifiers, suspending agents,surfactants, stabilizers, flavoring agents, excipients, vehicles,preservatives, binders and such, in a unit dose form required forgenerally accepted drug implementation. The amount of active ingredientin these preparations facilitates the acquisition of a suitable dosagewithin the indicated range.

Examples of additives that can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier such as oil can also beincluded in the above ingredients. Sterile composites for injections canbe formulated following normal drug implementations using vehicles suchas distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants such as D-sorbitol, D-mannnose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, and non-ionic surfactants such as Polysorbate 80(TM) and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer and may be formulated with a buffer such as phosphate bufferand sodium acetate buffer; a pain-killer, such as procainehydrochloride; a stabilizer, such as benzyl alcohol, phenol; and ananti-oxidant. The prepared injection may be filled into a suitableampule.

Methods well known to those skilled in the art may be used to administerthe inventive pharmaceutical to patients, for example as intraarterial,intravenous, percutaneous injections and also as intranasal,transbronchial, intramuscular, percutaneous, or oral administrations.The dosage and method of administration vary according to the bodyweight and age of the patient and the administration method; however,these can be routinely selected by one skilled in the art. If saidcompound is encodable by a DNA, the DNA can be inserted into a vectorfor gene therapy and the vector administered to perform the therapy. Thedosage and method of administration vary according to the body weight,age, and symptoms of the patient, but one skilled in the art can selectthem suitably.

The dose per time of a protein of this invention may vary depending onthe type of recipient, target organ, disease condition, andadministration method. For example, when injecting into a normal adult(body weight: 60 kg), it may be administered at about 100 μg to 20 mgper day.

The dose of a compound that binds to a protein of this invention, orthat of a compound that regulates the activity of a protein of thisinvention vary depending on the type of disease. For example, thecompound may be administered orally into a normal adult (body weight: 60kg) at about 0.1 to 100 mg per day, preferably at about 1.0 to 50 mg perday, and more preferably at about 1.0 to 20 mg per day.

When administered parenterally, the dose per time may vary depending onthe recipient, target organ, disease condition, and administrationmethod. For example, an appropriate dose can be, as an intravenousinjection into a normal adult (body weight: 60 kg), usually about 0.01to 30 mg per day, preferably about 0.1 to 20 mg per day, and morepreferably about 0.1 to 10 mg per day. For other animals, an amountconverted to dose per 60 kg body weight, or dose per body surface areamay be applied.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the MC-PIR1 cDNA (SEQ ID NO:1)and its deduced amino acid sequence (SEQ ID NO:2). Dotted line under thenucleotide sequence indicates the DNA fragment isolated by the SST-REXmethod. Poly-A signal is shown by the underline at the bottom. Thesignal sequence is indicated by lower case letters. Thickened underlineshows the transmembrane domain. The ITIM sequences are shown by doubledunderlines. Cysteines participating in S-S bond formation are boxed. Thebinding sequence for the asparagine-linked sugar chain is also boxed.

FIG. 2 shows the open-reading frame of MC-PIR2 (SEQ ID NO:3) and itsdeduced amino acid sequence (SEQ ID NO:4). The signal sequence is shownby lower case letters. The transmembrane domain is indicated bythickened underline. The lysine residue critical for binding to othercofactor molecule containing the ITAM sequence in the transmembranedomain is boxed. Cysteines participating in S-S bond formation are alsoboxed. The binding sequence for the asparagine-linked sugar chain isalso boxed.

FIG. 3 is a photograph showing the result of a PCR analysis of MC-PIR1and MC-PIR2 expression in different tissues. GAPDH was used as acontrol.

FIG. 4 is a photograph showing the result of an RT-PCR analysis ofMC-PIR1 and MC-PIR2 expression in different cell types. GAPDH was usedas a control.

FIG. 5 shows the result of a FACS analysis indicating the presence ofMC-PIR1 and MC-PIR2 on mast cell surface.

FIG. 6 is an electrophoresis photograph showing the result of tyrosinephosphorylation of a chimeric protein induced by cross-linking withanti-mouse IgG antibody.

FIG. 7 an electrophoresis photograph showing the result of complexformation with phosphotyrosine phosphatase SHP-1 and SHP-2, andphosphoinositide phosphatase SHIP.

FIG. 8 is an electrophoresis photograph showing that MC-PIR2 forms acomplex with ITAM-comprising signaling molecules DAP10, DAP12, and FcRγ.

FIG. 9 schematically shows the signal transduction of FcεRI and theinhibitory function of MC-PIR1.

BEST MODE FOR CARRYING OUT THE INVENTION

This invention will be explained in detail below with reference toExamples, but it is not to be construed as being limited thereto.

EXAMPLE 1 Construction of a cDNA Library and Screening

A cDNA library was constructed and expressed using the retrovirus vectorpMX-SST (Kojima T. and Kitamura T. Nature Biotechnol. 17: 487-490(1999)).

Poly-A(+) RNA was extracted from mast cells derived from mouse bonemarrow, stimulated with antigen using the Fast track 2.0 mRNA extractionkit (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sprotocol.

cDNA was synthesized from poly-A(+) RNA using the SuperScript ChoiceSystem (Invitrogen) and random hexamers, and inserted into the BstXIsite of pMX-SST vector using a BstXI adaptor (Invitrogen). To constructa SST-REX library, ligated DNA was amplified in DH10B cells (ElectroMax,Invitrogen), and library DNA was prepared using the Qiagen plasmid kit(Qiagen Inc., Valencia, Calif.). The size of the cDNA library was2.0×10⁶ clones.

High titer retroviruses presenting the SST-REX library was producedusing the Plat-E packaging cell line (Morita S. et al. Gene Therapy 7:1063-1066 (2000)), and used for infection of Ba/F3 cells as described.The day following infection, cells were washed three times, seeded in96-well multi titer plates (10³ cells/well), and clones were selected inthe absence of IL-3.

After twelve days, genomic DNA was extracted from factor independentBa/F3 clones, and subjected to genomic PCR using vector primers torecover inserted cDNA (GGGGGTGGACCATCCTCTA/SEQ ID NO: 5; andCGCGCAGCTGTAAACGGTAG/SEQ ID NO: 6). PCR was performed on GeneAmp PCRsystem 480 (Perkin Elmer, Norfolk, Conn.) using LA Taq polymerase(TaKaRa, Kyoto, Japan) in 30 cycles (each cycle consisting ofdenaturation at 98° C. for 20 seconds, followed by annealing andextension at 68° C. for 2 minutes). The obtained PCR fragment wasprocessed for sequencing using the Taq Dye-Terminator Cycle Sequencingkit (Applied Biosystems Inc., Foster City, Calif.), and analyzed usingan automated DNA sequencer (377 DNA analyzer; Applied Biosystems Inc.).

Differentiated cultured mast cells used in the experiment, which werederived from mouse bone marrow, were prepared as follows. Bone marrowcells were prepared from the thigh bone of CBA/JN mice, and cultured inRPMI 1640 supplemented with 10% FCS, 100 unit/ml of penicillin, 100μg/ml of streptomycin, and 10 ng/ml of mouse IL-3 at 37° C., 5% CO₂.Cells were passaged every couple of days at a density of 5×10⁵ cells,and maintained for four weeks to allow differentiation. Ba/F3 cells, amouse IL-3 dependent pro B-cell line, were cultured in RPMI 1640supplemented with 10% FCS, and 1 ng/ml of mouse IL-3 (R&D Systems).

Antigen stimulation of mast cells was performed as follows (Kawakami T.et al. J. Immunol. 148: 3513-3519 (1992)). Mast cells were challengedwith 0.5 μg/ml of anti-DNP-IgE antibody (Sigma) overnight, and the nextday, with 100 ng/ml of DNP-BSA (CosmoBio) for two hours.

EXAMPLE 2 Analysis of Isolated cDNA Clones

From the cDNA clones, a DNA fragment containing a signal sequence andencoding a single immunoglobulin domain was isolated.

A cDNA library was constructed using an oligo-dT primer. cDNA wassynthesized from poly-A(+) RNA using the SuperScript Choice System(Invitrogen) and oligo-dT primer, and inserted into the BstXI site ofpME18S vector using a BstXI adaptor (Invitrogen). To construct anoligo-dT cDNA library, ligated DNA was amplified in DH10B cells, andlibrary DNA was prepared using the Qiagen plasmid purification kit(Qiagen Inc.). The size of the cDNA library was 1.5×10⁸ clones.

Full-length cDNA was isolated by hybridization using RecA (DaiichiKagaku Yakuhin, Tokyo, Japan). Using the isolated cDNA fragment as atemplate, PCR reaction was performed to synthesize a probe ofapproximately 500 bp, in which biotin 21-dUTP was incorporated(Clontech). The probe was hybridized with the oligo-dT cDNA library inthe presence of RecA. DNA recovered using streptavidin magnetic beads(Promega) was amplified in DH10B cells (ElectroMax, Invitrogen), and E.coli clones were obtained. The E. coli clones were grown in a largescale, and DNA was prepared using the Qiagen plasmid kit (Qiagen Inc.).Purified DNA was processed for sequencing using the Taq Dye-TerminatorCycle Sequencing kit (Applied Biosystems Inc., Foster City, Calif.), andanalyzed on an automated DNA sequencer (377 DNA analyzer; AppliedBiosystems Inc.).

The obtained cDNA had a full length of 1752 bp, of which 957 nucleotidescomposed an open reading frame. The 3′-region, spanning 648 nucleotides,contained the poly-A-attaching signal. The deduced amino acid sequencecontained 318 residues, in which a signal sequence of 27 amino acids, anextracellular domain of 156 amino acids, a transmembrane domain of 23amino acids, and an intracellular domain of 112 amino acids were found.The extracellular domain had a single immunoglobulin domain of avariable type. The intracellular domain contained four ITIM-likesequences (FIG. 1). This gene was named MC-PIR1.

Genes having homology with this novel mouse gene are CMRF-35-H9(CMRF-35H) (Green B. J. et al. Int. Immunol. 10: 891-899 (1998);Accession No. AF020314), and IRp60 (Cantoni C. et al. Eur. J. Immunol.29: 3148-3159 (1999); Accession No. AJ224864). It is unknown, however,whether CMRF-35H or IRp60 is expressed in mast cells. According to thestructural similarities, these genes are considered to be the humanhomologues of MC-PIR1.

The MC-PIR1 sequence was used to search EMBL/GenBank/DDBJ DNA databases.As a result, a DNA sequence (Accession No. BC006801) havingapproximately 90% homology with the immunoglobulin domain of MC-PIR1 wasfound. The sequence information was used to design primers, and RT-PCRwas performed using total RNA prepared from mast cells. As a result,expression of a similar gene product was detected. Furthermore, the DNAfragment was recovered for sequence analysis.

As a result, a DNA having a sequence almost identical to that depositedin the DNA database (with two different bases at two sites compared tothe sequence of Accession No. BC006801, resulting in two amino acidsubstitutions) was obtained, and named MC-PIR2 (FIG. 2). Later, itturned out that MC-PIR2 encodes exactly the same protein as DIgR1 (LuoK. et al. Biochem. Biophys. Res. Commun. 287: 35-41(2001); Accession No.AY048685). The human gene CMRF-35A (Clark G J. et al. Tissue Antigens57: 415-423 (2001); Accession No. BC022279) is homologous to this mousegene. However it is not known whether or not DIgR1 or CMRF-35A isexpressed in mast cells, or what functions they have therein.

EXAMPLE 3 Expression Profile of MC-PIR1 and MC-PIR2

Expression profile of the genes was analyzed by PCR. A commercial DNA, acDNA synthesized from mRNA derived from different mouse tissues(Clontech), was used as a template for the PCR to amplify the DNAfragment. In addition, total RNA was prepared from a variety ofhematopoietic lineage cell lines using the Trizol reagent (Invitrogen),and used to prepare cDNA using a reverse transcriptase (Qiagen). ThecDNA was used as a template for PCR to amplify DNA fragment. AmplifiedDNA fragment was separated by electrophoresis on a 1% agarose gel.

Both MC-PIR1 and MC-PIR2 were expressed abundantly in the spleen andliver. For both, amplification of a specific DNA fragment was detectedin mouse bone marrow-derived cultured mast cells (BMMC). Amplificationof MC-PIR1 was not detected in other cell lines including Ba/F3, A20,EL4, CTLL-2, FDC-P1, L-G, 32Dc13, J774.1, and P815. Expression ofMC-PIR2 was detected in J774.1 and FDC-P1 as well (FIGS. 3 and 4). Theresult suggests that MC-PIR1 and MC-PIR2 is directly involved in theregulation of mast cell function.

EXAMPLE 4 Expression of MC-PIR1 and MC-PIR2 on the Cell Surface of MouseBone Marrow-Derived Mast Cells

Mast cells, prepared by the method in Example 1, were incubated withPE-labeled anti-CD117 monoclonal antibody (BD Pharmingen), and then withanti-MC-PIR1 mouse monoclonal antibody (custom made by R&D Systems) oranti-MC-PIR2 rabbit polyclonal antibody (custom made by Sigma Genosys).After washing, cells were incubated with FITC-labeled anti-mouseimmunoglobulin antibody (BD Pharmingen), or FITC-labeled anti-rabbitimmunoglobulin antibody (BD Pharmingen), respectively. After washing,cells were analyzed by FACS Calibur (Becton Dickinson).

About 90% to 95% of prepared cells were positive for CD117, and almostall the cells were induced to become mast cells. In CD117 positivecells, 90% were MC-PIR1 positive, and 25% was MC-PIR2 positive (FIG. 5).The result indicates that both MC-PIR1 and MC-PIR2 are present on themast cell surface.

EXAMPLE 5 Analysis of the Intracellular Domain of MC-PIR1

A chimeric gene between FcγRIIB and MC-PIR1 was constructed. A DNAfragment encoding the extracellular and transmembrane domains of FcγRIIBwas amplified by PCR. Similarly, a DNA fragment encoding theintracellular domain of MC-PIR1 was amplified by PCR. The two fragmentswere mixed and used as a template for PCR to amplify a DNA fragment andconstruct a chimeric gene. Then, a chimeric DNA fragment was digestedwith EcoRI and NotI, and inserted into pMX-IRES-puro vector to constructpMX-IRES-puro-Fc-PIR1.

A high titer stock of retroviruses presenting pMX-IRES-puro-Fc-PIR1 wasproduced in the Plat-E packaging cell line and used to infect IIA1.6cells, an FcγRIIB deficient cell line, as described (Jones B. et al. J.Immunol. 136: 348-356 (1986)). A day after infection, the medium wassupplemented with 1 μg/ml of puromycin (Clontech), and the culture wascontinued for an additional week to obtain a cell line expressing thechimeric gene.

The cell line expressing the chimeric gene was incubated with ananti-mouse IgG antibody (Zymed) to cross-link B-cell receptor and thechimeric gene product expressed on the cell surface, harvested over atime course, and lysed in cell lysis buffer. 2.4G monoclonal antibody(Becton Dickinson) was added to the cell lysate and anti-rat IgGantibody-Sepharose beads, and an immune complex was precipitated. Thepellet was treated with peptide N-glycosidase F (Daiichi KagakuYakuhin), and subjected to electrophoresis on a 10% polyacrylamide gel(PAGE).

The immune complex separated by PAGE was electrically transferred ontoan Immobilon-P membrane (Millipore). The membrane was blocked withbuffer containing 10% FCS, and then incubated successively with the 4G10monoclonal antibody (UpState Biotechnology) and HRP-conjugatedanti-mouse immunoglobulin antibody (Sigma). The signal was detectedusing chemiluminescence reagents (Pharmacia).

Similarly, cells expressing the chimeric gene were cross-linked, and amembrane was prepared as described. The membrane was incubated with ananti-SHP-1 antibody (Santa Cruz), anti-SHP-2 antibody (Santa Cruz), oranti-SHIP antibody. The detection was performed as described above.

Tyrosine phosphorylation of the chimeric protein was detected 0.5minutes after cross-linking. Furthermore, an immune complex containingthe chimeric protein was found containing SHP-1, SHP-2, and SHIP. Thecomplex formation was dependent on tyrosine phosphorylation of thechimeric protein (FIGS. 6 and 7).

EXAMPLE 6 Complex Formation Between MC-PIR2 and ITAM-ComprisingSignaling Molecules

MC-PIR2 was amplified by PCR using a primer attached with an HA tag atthe C-terminal end. The obtained fragment was digested with EcoRI andNotI, and inserted into the pMKIT vector to construct pMKIT-MC-PIR2—HA.

The DNA fragment encoding a mature protein of ITAM sequence-comprisingproteins DAP10, DAP12, or FcRγ was amplified by PCR. The amplified DNAfragment was digested with HindIII and NotI, and inserted into thedownstream of the FLAG tag in the pMKIT-FLAG vector.

PMKIT-MC-PIR2-HA and either the pMKIT mock vector, FLAG-DAP10 vector,FLAG-DAP12 vector, or FLAG-FcRγ vector were transfected into COS1 cells.After two days, cells were harvested, and lysed in a cell lysis buffer.An anti-HA monoclonal antibody (12CA5, Roche Diagnostics), and ProteinA-Sepharose beads were added to the cell lysate, and an immune complexwas precipitated. The pellet was subjected to electrophoresis on a 15%polyacrylamide gel (PAGE). The immune complex separated by PAGE waselectrically transferred onto an Immobilon-P membrane (Millipore). Themembrane was blocked with a buffer containing 10% FCS, and thenincubated successively with an anti-FLAG-M2 monoclonal antibody (Sigma),and an HRP-conjugated anti-mouse immunoglobulin antibody (Sigma). Thesignal was detected using chemiluminescence reagents (Pharmacia).

In the lane containing the lysate of cells transfected with MC-PIR2 andpMKIT mock vector, no band was detected with the anti-FLAG antibody. Incontrast, the same anti-FLAG antibody detected a band in the lanescontaining the lysates of cells transfected with MC-PIR2 and FLAG-DAP10,or FLAG-DAP12, or FLAG-FcRγ. Thus, MC-PIR2 was shown to interact withITAM-comprising signaling molecules, DAP10 (Wu J. et al. Science 285:730-732 (1999)), DAP12 (Lanier L. L. et al. Nature 391: 03-707(1998)),or FcRγ (Vivier E. et al. Int. Immunol. 4: 313-1323 (1992)) (FIG. 8).The result suggests that MC-PIR2 participates in the regulation ofactivation signal transduction.

INDUSTRIAL APPLICABILITY

The present invention provides genes encoding novel mast cell-derivedmembrane proteins considered to be involved in the regulation of signaltransduction in mast cells. These gene products are expected to inhibitor activate the signal transduction in mast cells following antigenstimulation through the following working hypothesis based on theirexpression profile and their ability to bind to proteins participatingin signal transduction.

When FcεRI is cross-linked by IgE and antigens in mast cells, theactivity of protein kinases increases in the cells, followed by increasein phosphatidylinositol turnover, which then results in an increase ofintracellular calcium ion concentration and induction of degranulation.In addition, because PI3K is also activated, the level of PIP3 isincreased on the plasma membrane, which leads to activation of Btk andthe like (Kawakami Y. et al. Mol. Cell. Biol. 14: 5108-5113 (1994)). Theinhibitory signal transduction pathways generally known are the SHIPdependent pathway (Muta T. et al. Nature 368: 70-73 (1994)) observed forFcγRIIB, and the SHP dependent pathway mediated by tyrosine phosphatases(Binstadt B. A. et al. Immunity 5: 629-638 (1996)). Because MC-PIR1 iscapable of activating both pathways together, it is expected to inhibitthe signaling from FcεRI more strongly (FIG. 9).

On the other hand, MC-PIR2 is capable of making a complex withITAM-comprising signaling molecules DAP10, DAP12, and FcRγ, and thus,are expected to induce activation through kinases such as the Src familykinases, or PI3 kinase, or the like (Wu J. et al. Science 285: 730-732(1999); Lanier L. L. et al. Nature 391: 703-707 (1998); Vivier E. et al.Int. Immunol. 4:1313-1323 (1992)). Therefore, inhibition of complexformation between MC-PIR2 and these ITAM-comprising signaling moleculesis expected to inhibit the transduction of the activation signal, forexample. Thus, it is possible that MC-PIR2 itself is a target moleculeof suppressors of mast cell activation signal transduction.

Products of MC-PIR1 and MC-PIR2 genes, and their human homologues willbe useful for screening natural ligands, compounds mimicking theireffects, or antibodies. Such ligands, compounds, or antibodies obtainedby the above screenings, could inhibit mast cell activation signaltransduction, and can be used as anti-allergy agents that functionthrough a novel mechanism.

1. An isolated DNA according to any one of the following (a) to (c): (a)a DNA encoding a protein comprising the amino acid sequence of SEQ IDNO:2, (b) a DNA comprising the coding region of the nucleotide sequenceof SEQ ID NO:1, (c) a DNA encoding a protein comprising an amino acidsequence in which up to 30 amino acids in the amino acid sequence of SEQID NO:2 have been replaced, deleted, inserted, and/or added, wherein theDNA encodes a protein capable of binding to a protein selected from thegroup consisting of SHP-1 protein, SHP-2 protein, and SHIP protein.
 2. Avector into which the DNA of claim 1 has been inserted.
 3. A host cellcarrying the DNA of claim 1 or a vector into which the DNA of claim 1has been inserted.
 4. A method for producing a protein which comprisesthe steps of culturing the host cell of claim 3, and recovering theexpressed protein from said host cell or the culture supernatantthereof.
 5. An isolated polynucleotide comprising a segment of SEQ IDNO:1 or the complementary strand thereof, the segment being at least 15nucleotides in length.
 6. The DNA of claim 1, wherein the DNA encodes aprotein comprising an amino acid sequence in which up to ten amino acidsin the amino acid sequence of SEQ ID NO:2 have been replaced, deleted,inserted, and/or added.
 7. The DNA of claim 1, wherein the DNA encodes aprotein comprising an amino acid sequence in which up to five aminoacids in the amino acid sequence of SEQ ID NO:2 have been replaced,deleted, inserted, and/or added.
 8. An isolated DNA that encodes aprotein that is 85% or more identical to SEQ ID NO:2, wherein theprotein is capable of binding to a protein selected from the groupconsisting of SHP-1 protein, SHP-2 protein, and SHIP protein.
 9. The DNAof claim 8, wherein the DNA encodes a protein that is 95% or moreidentical to SEQ ID NO:2.
 10. The DNA of claim 8, wherein the DNAencodes a protein that is 96% or more identical to SEQ ID NO:2.
 11. TheDNA of claim 8, wherein the DNA encodes a protein that is 97% or moreidentical to SEQ ID NO:2.
 12. The DNA of claim 8, wherein the DNAencodes a protein that is 98% or more identical to SEQ ID NO:2.
 13. TheDNA of claim 8, wherein the DNA encodes a protein that is 99% or moreidentical to SEQ ID NO:2.
 14. The DNA of claim 1, wherein the DNAencodes a protein comprising the amino acid sequence of SEQ ID NO:2. 15.The DNA of claim 1, wherein the DNA comprises the coding region of thenucleotide sequence of SEQ ID NO:1.
 16. The DNA of claim 1, wherein theDNA encodes a protein consisting of the amino acid sequence of SEQ IDNO:2.
 17. The DNA of claim 1, wherein the DNA consists of the codingregion of the nucleotide sequence of SEQ ID NO:1.